Effect of a mammary-derived growth inhibitor on the expression of the oncogenes c-fos, c-myc and c-ras.

A mammary-derived growth inhibitor (MDGI) inhibits the resumption of growth of stationary Ehrlich ascites carcinoma (EAC) cells in vitro. The present study shows that the resumption of growth is accompanied by a rapid increase of the steady state mRNA level of the proto-oncogenes c-fos, c-myc and c-ras, which is reduced by MDGI. EAC cells from the exponential growth phase insensitive to MDGI did not show a reduced RNA expression. The effect of MDGI represents a novel activity at the level of gene expression and suggests a link to exist between growth inhibition and the reduction of c-fos, c-myc and c-ras expression.

Altered growth control by natural growth inhibitors in the mutant HD33 substrain of the Ehrlich-Lettré ascites mammary carcinoma.

The mutant substrain HD33 of the Ehrlich-Lettré ascites mammary carcinoma (EAC) was found to be altered in its ability to respond to and to produce natural growth inhibitors. Cells of this substrain did not respond to (1) a highly purified growth inhibitor from bovine mammary gland, inhibiting the proliferation of two strains of the original EAC already at concentrations of 0.5-2.0 ng/ml, (2) inhibitory activities found in the ascites fluid of these sensitive strains and partially purified. However, from the ascites fluid of the mutant substrain an inhibitory activity was partially purified, which was inhibitory towards this substrain. It also inhibited the cells from an original strain, although less effectively. This new inhibitory activity is similar in its properties to that of the others investigated in that its mol. wt is between 10,000 and 50,000 D, it is heat labile and its activity can be overcome by insulin, epidermal growth factor and 2'-deoxycytidine, the dose-response curve levels off at 35-45% inhibition. The results demonstrate that the regulation of cell proliferation by endogenous (autocrine) inhibitors can be altered by mutagen treatment.

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy.

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.

Fetal calf serum and epidermal growth factor prevent the inhibitory action of a chalone-like growth inhibitor for Ehrlich ascites mammary carcinoma cells in vitro.

A growth inhibitor for the Ehrlich ascites mammary carcinoma, purified from bovine mammary gland, is antagonized by very low concentrations of epidermal growth factor (2-16 X 10(-10) M) or fetal calf serum (0.001-0.1%). Thus, epidermal growth factor and the compounds in fetal calf serum have the same effect as earlier described for insulin and proinsulin [1-3].

Is ribonucleotide reductase in Ehrlich ascites mammary tumour cells the target of a growth inhibitor purified from bovine mammary gland?

2'-deoxycytidine (dCyd), 2'-deoxyuridine (dUrd) and cytidine (Cyd) were found to relieve the inhibitory action by a factor, highly purified from bovine mammary gland, on the resumption of growth in vitro of stationary Ehrlich ascites mammary tumour cells. As described earlier, this effect is also achieved by insulin, proinsulin and epidermal growth factor and fetal calf serum. The effect of purine-2'-deoxyribonucleosides could not be properly assessed, because they were inhibitory themselves. Purine ribonucleosides as well as uridine and uracil were inactive. Because of the inactivity of the latter two compounds, the effect is attributed to an influence of the factor on the ribonucleotide reductase (RR) (and not on the de novo pyrimidine synthesis). The active nucleosides need not be present for the whole incubation time of 24 h; when treated for 4 h with the nucleosides, which are then removed before the addition of the factor, cells become insensitive against the latter. Again a similar effect has been described earlier for serum and insulin. The inhibitor of RR, hydroxyurea, prevents this specific effects of serum and (partially) insulin as well that of dCyd, also suggesting the involvement of RR in the factor action.

Purification of a chalone-like inhibitor for Ehrlich ascites mammary carcinoma cells from bovine mammary gland.

A factor which inhibits the resumption of proliferation of stationary Ehrlich ascites mammary carcinoma (EAC) cells in vitro was isolated from the normal, lactating bovine mammary gland. After fractionation of the 50,000 g supernatant of the tissue homogenate by ammonium sulfate precipitation and gel filtration, it was further purified 2600-fold by ultrafiltration and lectin affinity chromatography. Polyacrylamide electrophoresis revealed one band containing all the activity. SDS gel electrophoresis showed a main band corresponding to a protein with a molecular weight of about 15,000 dalton. At this state of purity the factor concentration for half-maximal inhibition of proliferation was 12 ng/ml or 8 X 10(-10) M. The effects of factor concentrations of 4 X 10(-9) or 4 X 10(-8) M are abolished by 5 X 10(-9) M insulin almost completely or to 70%.

Aspects of chalone action.

The following aspects of action of a chalone-like factor for the Ehrlich ascites mammary carcinoma and of the granulocytic chalone are discussed: 1) Dependence on the state of proliferation. Rapidly growing cells respond poorly or not at all. 2) Antagonism with growth factors. The factor for the Ehrlich ascites mammary carcinoma is antagonized by physiological concentrations of insulin or by proinsulin. The granulocytic chalone is antagonized by colony stimulating factor. 3) Influence on cell cycle progression. Attempts and problems to analyse this influence for the factor for the Ehrlich ascites mammary carcinoma by flow cytometry are described.

[Mathematical methods in flow cytometry: the problem of evaluating DNA histograms of partially synchronous cell populations]. / Mathematische Methoden in der Impulszytophotometrie: Zum Problem der Auswertung von DNS-Histogrammen teilsynchronisierter Zellpopulationen.

There is presented a procedure for determining the phase fractions in a cell population (i.e. the fractions of the G1-, S- and G2 + M-cells) from the corresponding DNA histogram obtained by flow cytometry. The evaluation procedure can regard arbitrary apriori information about the model parameters. It is based on a mathematical model of the DNA distribution for a growing cell population which contains the phase fractions and some further values as parameters. The parameter estimations rest on the Maximum-Likelihood-Principle by which the parameters of the theoretical model must be determined in such a way that the measured histogram corresponds to a maximum probability. As a consequence of the model flexibility especially the S-phase fraction which can be determined independently by labeling techniques, can be regarded as a fixed predetermined value. Such apriori information enhances the preciseness of the parameter estimation. The applications have shown that the determination of the S-phase fraction by means of flow cytometry can be very unprecise especially under partial synchronization. For the application of the evaluation programs (programming language ALGOL) the histograms must be free of cell detritus and clumping.

On a "chalone"-like factor for Ehrlich ascites mammary carcinoma.

In an aqueous ultrafiltrate (10 000--50 000) prepared from Ehrlich ascites mammary carcinoma (EAC) cells, ascites fluid and bovine mammary gland, a new factor was obtained which is involved in the growth regulating system of EAC cells as shown by the following facts: 1) It reversibly inhibits the proliferation of EAC cells in a 24 h suspension culture, depending on their proliferative state. Thus, stationary cells from the plateau phase of growth in vivo are prevented from resuming growth in vitro, while cells taken from the active phase of in vivo growth are not inhibited under the same conditions. Likewise, stationary cells do not respond when incubated in serum before the addition of the factor (depending on serum concentrations and time). The dose-response curve levels off at higher factor concentrations so that the maximal inhibition is about 50--65% (explained with different sensitivities of the cells in the assay system). The time course of the inhibition as well as preliminary data from flow cytophotometry and labeling with tritiated thymidine indicate an interference with the progression of G1-phase cells into the S-phase. 2) The activity of the factor is counteracted by insulin and proinsulin, both known as growth factors. The insulin concentration needed is dependent on the factor concentration; an almost maximal inhibition can be prevented by physiological concentrations of insulin. The activity can be destroyed by heat and trypsin and differs also in other properties from that of polyamines. The factor could not be detected in lung, liver, spleen, kidney, heart and L 1210 ascites fluid of the mouse or in bovine malignant lymph nodes, thymus, kidney or liver. The factor was purified from a homogenate of bovine mammary gland by ultrafiltration and affinity chromatography on sepharose-bound wheat lectin. Polyacrylamide electrophoresis showed about 5 bands of which one contained the activity (in some experiments overlapping with a second one). In this way a 800fold purification (starting from the ultrafiltrate) could be obtained. The overall purification (relative to the centrifuged homogenate) can be calculated to be more than 100 000 fold.

Chalone-like inhibition of Ehrlich ascites cell proliferation in vitro by an ultrafiltrate obtained from the ascitic fluid.

An aqueous ultrafiltrate (10 000-50 000 dalton) prepared from the cell-free ascitic fluid of mice bearing Ehrlich ascites tumour (EAT) in the plateau phase of growth (12-16 days after transplantation) was investigated with regard to its inhibitory effects on the proliferation of EAT cells in a 24-hr suspension culture. The following results were obtained: (1) The in vitro proliferation of cells obtained from the plateau phase of in vivo growth was reversibly inhibited. (2) The dose-response curves show a plateau with a maximum inhibition of about 50%, which suggests that not all cells can be affected. (3) Young cells (4-6 days after transplantation) were not inhibited. (4) Preincubation of plateau phase cells in the culture medium before treatment abolishes the inhibitory effect of the ultrafiltrate. This effect of preincubation is dependent on time and serum concentration. It provides the possibility to differentiate between true "chalone-like" and cytotoxic effects. (5) the inhibitory properties of the ultrafiltrate are destroyed by heating or trypsin treatment. (6) Extracts prepared in the same way from ascitic fluid of mice bearing lymphocytic leukemia L1210 do not inhibit the proliferation of EAT cells. Corresponding extracts from ascitic fluid of mice bearing myelocytic leukemia YM were found to be inhibitory; however, the inhibitory effect was also found on preincubated cells and is therefore considered to be due to an unspecific cytotoxicity. In conclusion, evidence was obtained for a factor from the ascitic fluid of mice bearing EAT, which prevents EAT cells from entering the proliferating state.